human LPA1 (Kb = 6.9 nM), mouse LPA (Kb = 4.0 nM)[1]

In CHO cells overexpressing LPA1, BMS-986278 is a high-affinity LPA1 antagonist with Kbs of 6.9 nM for human and 4.0 nM for mouse LPA1 [1]. With a Kb of 5.8 nM, BMS-986278 inhibits the calcium flux in normal human lung fibroblasts when stimulated by lysophosphatidic acid (LPA) [1].

BMS-986278 (0.1-10 mg/kg; single oral dose) totally suppresses LPA-stimulated systemic histamine release in CD1 mice in a concentration-dependent manner [1]. BMS-986278 (3-30 mg/kg; orally twice daily for 14 days) decreases bleomycin-induced collagen deposition/pulmonary fibrosis in rats [1]. Pharmacokinetics of BMS-986278 in preclinical species [1] Plasma clearance ((mL/min)/kg) Vss (L/kg) Oral bioavailability (%) T1/2 (h) Plasma protein binding (% free) Mouse 37 5.5 70 2.5 31.4 Rat 15 3.5 100 4.5 12.6 Monkey 2.0 1.6 79 11 0.8

In Vitro Biological Assays[1]
The effectiveness of compounds as LPA1 inhibitors can be determined in an LPA1 functional antagonist assay as follows:
CHO cells overexpressing either human LPA1, mouse LPA1 receptors, or normal human lung fibroblasts (NHLF, Lonza, MD, USA) were plated at 20,000 or 10,000 cells per well respectively in 0.02 mL volume in poly-d-lysine-coated 384-well microplates and incubated overnight at 37° with 5% CO2. The next day, cells were loaded with calcium indicator dye for 30 min at 37 °C and then equilibrated to RT for 30 min before assay. Test compounds solubilized and serially diluted in 100% DMSO were transferred to 384-well nonbinding surface plates using a Labcyte Echo acoustic dispenser and further diluted with assay buffer (1× HBSS (Hanks balanced salt solution) with calcium/magnesium, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer, and 0.1% fatty acid-free BSA) to 0.5% DMSO before addition to the cells at final concentrations ranging from 0.08 nM to 5 μM. Cells were subsequently incubated for 20 min at RT before stimulation with LPA at final concentrations of 10 nM (EC50). Inhibition of LPA-stimulated calcium flux was monitored for 90 s after the addition of LPA. IC50 values (defined as the concentration of test compound that inhibits 50% of the LPA response) were determined, using the four-parameter logistic equation. The functional affinity Kb was calculated from the IC50 values using the functional Cheng–Prusoff equation:
where [A] is the concentration of LPA used in the antagonist assay, EC50 is the concentration of LPA that causes 50% maximal activity of the LPA agonist control curve, and h is the slope value obtained from the fit of the agonist data for LPA.

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In Vitro Metabolite Elucidation[1]
Compound 33 (10 μM) was incubated with liver microsomes (1 mg/mL) from mouse and human in the presence of NADPH (1 mM) and GSH (1 mM) at 37 °C for 0, 20, and 60 min. The samples were deproteinated by adding an equal volume of acetonitrile, followed by centrifugation at 21000g for 10 min. The supernatants were concentrated to half-volume and injected into an LC/UV/MS system. The metabolism of 33 was evaluated in hepatocytes from human, monkey, dog, rat, and mouse. Cryopreserved hepatocytes from mouse, rat, dog, monkey, and human were suspended in a Krebs–Henseleit buffer (∼1 × 106 cells/mL) and incubated with 33 (10 μM) for 2 h at 37 °C in a humidified CO2 (5%) incubator. After the reaction, samples were processed by the same method described above. The metabolism of 25 was evaluated in the same manner.

Serum Protein Binding Assay[1]
Compound 33 was tested in a panel of multispecies serum protein binding assays to determine the extent to which this compound binds to serum proteins in various species. In a single test occasion, six independent samples of 33 were assayed in triplicate by combining with serum from an individual species of interest (human, rat, mouse, or cynomolgus monkey serum) to achieve final concentration of 10 μM. Dialysis was performed for 5 h at 37 °C, in a 10% CO2 atmosphere against sodium phosphate buffer using the two-chamber Rapid Equilibrium Dialysis (RED) Assay Plates from Thermo Fisher. Assay samples from buffer and serum chambers were collected at time zero (T0[serum] and T0[buffer]) and at 5 h postincubation (postequilibration, T5 h[serum] and T5 h[buffer]). Samples were analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) to assess the fraction of compound (percentage) free to diffuse and equilibrate between the buffer and serum chambers in the dialysis device. Prior to LC-MS/MS analysis, assay samples were diluted with either buffer or serum to result in the same final serum concentration in each sample. Subsequently, these samples were extracted by protein precipitation in acetonitrile containing analytical internal standards. Samples were analyzed by LC-MS/MS, and relative amounts of 33 were determined by calculating the peak area ratio of 33 to internal standard in each sample. Results were expressed as the percent of 33 free (unbound), percent bound, and percent recovered after incubation. The serum protein binding results of other compounds were obtained in an analogous fashion.

Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat (10 weeks) given bleomycin [1]
Doses: 3, 10 and 30 mg/kg
Doses: Orally, twice (two times) daily for 14 days
Experimental Results: The percentage of lung section fibrosis area was Dramatically diminished in the 3 mg/kg (48%) and 10 mg/kg (56%) dose groups.

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LPA Challenge with Plasma Histamine Evaluation (Acute Pharmacodynamic Assay)[1]
The effect of compounds on LPA-stimulated histamine release was assessed in 10 week old female (Hsd:ICR) CD-1 mice. (9c,19) Compounds were administered orally (PO) in a vehicle containing 40% PEG400, 10% cremophor, and 50% phosphate buffered saline (PBS) at a dose volume of 10 mL/kg. Two hours after dosing, animals (each dose group = 10 mice) were challenged with a single tail vein injection of PBS or PBS plus 300 μg of lysophosphatidic acid (LPA, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate [sodium salt]). LPA was prepared in 0.1% fatty acid free BSA/PBS at 2 μg/μL. Two minutes after the LPA injection, mice were sacrificed via decapitation and blood was collected in ethylenediaminetetraacetic acid (EDTA)-coated tubes, spun down (800× gravity, 10 min). The plasma was separated and stored frozen at −80 °C until assayed for compound concentrations and histamine levels. Histamine levels were determined by a competitive enzyme-linked immunosorbent assay (ELISA) as per manufacturer’s instruction (Histamine EIA, Oxford Biomedical Research). The percentage histamine release was calculated by designating the mean of the PBS plus the LPA group as 100% histamine release, and the PBS group minus LPA as 0% histamine release. [1]

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Rat Bleomycin Chronic Efficacy Studies[1]
Compounds were assessed in a 21 day bleomycin-induced lung fibrosis model modified from the literature. (9c) Male Sprague-Dawley rats, aged 10 weeks, were purchased from Charles River Laboratories. After a 10 day acclimation, rats were anesthetized with isoflurane (4% in 100% O2) and administered bleomycin (3.5 U/kg, delivered in a volume of 2 μL/g sterile saline) via oropharyngeal instillation, using a method modified from the literature. (20a) Rats were subsequently returned to their cages until they fully recovered from anesthesia and were monitored daily for the duration of the experiment. On day 7 post-bleomycin instillation, animals were randomized into groups of 10–12, on the basis of their body weight loss since instillation, and compound dosing was initiated on day 8. On day 21, rats were euthanized, left lung lobes were removed, inflated, fixed in formalin, and processed into paraffin blocks, which were sectioned and stained with Picrosirius Red (PSR) (20c) or Masson’s trichrome (MT). (20d) Stained slides were digitized and were analyzed via HALO software a semiautomated histological scoring system. PSR staining was scored by analyzing the entire lung section for the proportion of tissue stained red (%PSR positive), indicating the presence of collagen. MT stained slides were analyzed for areas of increased optical density (%fibrotic area), as a surrogate for the proportion of lung displaying bleomycin-induced injury/inflammation. [1]

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Mouse Pharmacokinetic Studies[1]
The mouse pharmacokinetics study of BMS-986278 (compound 33) was carried out in male CD-1 mice. Animals (N = 4) were fasted overnight (for oral administration only) and received 33 either as an IV bolus dose (1 mg/kg) via a tail vein or by oral gavage (3 mg/kg). Blood samples (∼0.2 mL) were obtained by retro-orbital bleeding at 0.05 (or 0.25 for oral), 0.5, 1, 4, 7, and 24 h postdose. Within each group, half of the animals were bled at 0.05 (or 0.25 for oral), 0.5, 1, 3, 5, 7, and 24 h. Blood samples were allowed to coagulate and centrifuged at 4 °C (1500–2000 × gravity) to obtain serum. Serum samples were stored at −20 °C until analysis by LC-MS/MS (Characterization of 25). Compound 33 was formulated in 5% dimethyl acetamide and 95% tris buffer for IV administration. For PO administration at 3 mg/kg, 33 was formulated as a solution in 10% cremophor-EL, 40% PEG400, and 50% aqueous phosphate buffer. Blood samples were collected; serum was prepared and stored as described above until analysis by LC-MS/MS. A similar protocol was used for the mouse PK study of 25.[1]

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Rat Pharmacokinetics Studies[1]
The rat pharmacokinetics study of BMS-986278 (compound 33) was carried out in male Sprague-Dawley rats (300–350 g). Rats were fasted overnight for PO administration only. Blood samples (∼0.3 mL) were collected from the jugular vein into tripotassium ethylenediaminetetraacetic acid (K3EDTA)-containing tubes at 0.17 (IV only), 0.25, 0.5, 0.75, 1, 2, 3, 5, 7, and 24 h postdose and then centrifuged at 4 °C (1500–2000g) to obtain plasma, which was stored at −20 °C until analysis by LC-MS/MS (Characterization of 25). A similar protocol was used in the rat PK studies of 25 and the other compounds for which rat PK data are described in this paper.[1]

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Cynomolgus Monkey Pharmacokinetic Studies[1]
The monkey pharmacokinetics profile of BMS-986278 (compound 33) was evaluated in a crossover-design study in male cynomolgus monkeys. Following an overnight fast, 3 animals (5–8 kg) received 33 by IV infusion (1 mg/kg over 10 min) via a femoral vein and by oral gavage (5 mg/kg), with a 1 week washout between treatments. Serial blood samples (∼0.3 mL) were collected from a femoral artery into K3EDTA-containing tubes predose and at 0.17 (IV only), 0.25, 0.5, 0.75, 1, 2, 3, 5, 7, and 24 h postdose and centrifuged at 4 °C (1500–2000g) to obtain plasma. Samples were stored at −20 °C until analysis by LC-MS/MS (Characterization of 25). A similar protocol was followed for the cynomolgus monkey PK study of 25.

[1]. Discovery of an Oxycyclohexyl Acid Lysophosphatidic Acid Receptor 1 (LPA1) Antagonist BMS-986278 for the Treatment of Pulmonary Fibrotic Diseases. J Med Chem. 2021 Nov 11;64(21):15549-15581.

The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).[1]

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Compound 33 is a potent LPA1 antagonist in vitro (LPA1Kb = 6.9 nM in CHO cells overexpressing human LPA1 and LPA1Kb = 5.9 nM in normal human lung fibroblasts). In a 21 day study (in therapeutic mode) in the chronic rat bleomycin lung fibrosis model, 33 demonstrated robust antifibrotic efficacy; it significantly decreased lung fibrosis area in the bleomycin-treated rats at the 3 and 10 mg/kg BID doses, as determined by histological analysis (Masson’s trichrome staining) at study termination. Overall, 33 has an excellent in vitro liability profile, with (1) no inhibition of any key CYP450 enzymes or ion channels (hERG, sodium, and L-type calcium) that would represent safety concerns and (2) no induction of CYP450 enzymes in human hepatocytes. Importantly, 33 has negligible activity at key hepatic transporters, especially the hepatobiliary bile acid transporters such as BSEP, MDR3, MRP3, and MRP4. An excellent pharmacokinetic profile for 33 was observed in three preclinical species (mouse, rat, and monkey). The discovery of 33 accomplished our objective of significantly improving upon 1 (hepatic/DDI transporter inhibition, aqueous solubility and human hepatocyte metabolic stability), thus validating our design strategy of reducing cLogP and increasing C(sp3) fraction. In conclusion, 33 is a novel LPA1 receptor antagonist with an excellent biological and liability profile which has promise for the treatment of fibrotic diseases. Compound 33 has been advanced into clinical studies; a single-ascending dose and a multiple-ascending dose study have both been completed. A Phase 2 clinical trial with 33 in patients with IPF and PF-ILD has been initiated (ClinicalTrials.gov Identifier NCT04308681).[1]

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BMS-986278 (33) has to date completed several early stage clinical trials, including a single ascending dose (SAD) study and a 14 day multiple ascending dose (MAD) study in normal healthy volunteers. Compound 33 was generally well tolerated at all doses up to 125 mg BID. Linear and dose-proportional pharmacokinetics was observed for 33 with a very low degree of accumulation following either once daily or twice daily dosing. At the highest dose of 125 mg twice daily, the daily exposure of 33 as defined by AUC was ∼17-fold higher than the AUC achieved with the 600 mg twice daily dose of 1. In terms of pharmacokinetics in normal healthy volunteers, 33 was rapidly absorbed orally with a Tmax of 1.5 h and a half-life of ∼12 h. A global Phase 2 clinical trial in patients with IPF or PF-ILD has been initiated with 33 (ClinicalTrials.gov Identifier NCT04308681).[1]

C22H31N5O5

445.5120

445.23

C, 59.31; H, 7.01; N, 15.72; O, 17.96

2170126-74-4

132232205

White to light yellow solid powder

2.1

1

8

9

32

638

2

O(C1C([H])=C([H])C(C2=C(C([H])([H])OC(N(C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[H])=O)N(C([H])([H])[H])N=N2)=NC=1C([H])([H])[H])[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])[C@]([H])(C(=O)O[H])C1([H])[H]

UEUNDURNLYLSNB-HOTGVXAUSA-N

InChI=1S/C22H31N5O5/c1-5-11-26(3)22(30)31-13-18-20(24-25-27(18)4)17-9-10-19(14(2)23-17)32-16-8-6-7-15(12-16)21(28)29/h9-10,15-16H,5-8,11-13H2,1-4H3,(H,28,29)/t15-,16-/m0/s1

(1S,3S)-3-[2-methyl-6-[1-methyl-5-[[methyl(propyl)carbamoyl]oxymethyl]triazol-4-yl]pyridin-3-yl]oxycyclohexane-1-carboxylic acid

BMS-986278; 2170126-74-4; Admilparant; 4UN9AOU6G8; UNII-4UN9AOU6G8; BMS986278; (1S,3S)-3-((2-Methyl-6-(1-methyl-5-(((methyl(propyl)carbamoyl)oxy)methyl)-1H-1,2,3-triazol-4-yl)pyridin-3-yl)oxy)cyclohexane-1-carboxylic acid; Cyclohexanecarboxylic acid, 3-((2-methyl-6-(1-methyl-5-((((methylpropylamino)carbonyl)oxy)methyl)-1H-1,2,3-triazol-4-yl)-3-pyridinyl)oxy)-, (1S,3S)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~224.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)